Releasable linkage and compositions containing same

ABSTRACT

A compound comprised of a hydrophilic polymer covalently yet reversibly linked to a amine-containing ligand through a dithiobenzyl linkage is described.

[0001] This application is a continuation of U.S. application Ser. No.09/982,336 filed Oct. 12, 2001, now pending; which is a continuation ofU.S. application Ser. No. 09/556,056 filed Apr. 21, 2000, now U.S. Pat.No. 6,342,244; which claims the benefit of U.S. Provisional ApplicationNo. 60/130,897 filed Apr. 23, 1999, now abandoned; all of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to a compound comprised of ahydrophilic polymer, such as polyethyleneglycol, cleavably linked to anamine-containing ligand, which in preferred embodiments can be anamine-containing lipid, drug or protein. The compounds are cleavableunder mild thiolytic conditions to regenerate the amine-containingligand in its original form.

BACKGROUND OF THE INVENTION

[0003] Hydrophilic polymers, such as polyethylene glycol (PEG), havebeen used for modification of various substrates, such as polypeptides,drugs and liposomes, in order to reduce immunogenicity of the substrateand/or to improve its blood circulation lifetime.

[0004] For example, parenterally administered proteins can beimmunogenic and may have a short pharmacological half-life. Proteins canalso be relatively water insoluble. Consequently, it can be difficult toachieve therapeutically useful blood levels of the proteins in patients.Conjugation of PEG to proteins has been described as an approach toovercoming these difficulties. Davis et al. in U.S. Pat. No. 4,179,337disclose conjugating PEG to proteins such as enzymes and insulin to formPEG-protein conjugates having less immunogenicity yet which retain asubstantial proportion of physiological activity. Veronese et al.(Applied Biochem. and Biotech, 11:141-152 (1985)) disclose activatingpolyethylene glycols with phenyl chloroformates to modify a ribonucleaseand a superoxide dimutase. Katre et al. in U.S. Pat. Nos. 4,766,106 and4,917,888 disclose solubilizing proteins by polymer conjugation. PEG andother polymers are conjugated to recombinant proteins to reduceimmunogenicity and increase half-life. (Nitecki et al., U.S. Pat. No.4,902,502; Enzon, Inc., PCT/US90/02133). Garman (U.S. Pat. No.4,935,465) describes proteins modified with a water soluble polymerjoined to the protein through a reversible linking group.

[0005] However, PEG-protein conjugates described to date suffer fromseveral disadvantages. For example, modification of the protein with PEGoften inactivates the protein so that the resulting conjugate has poorbiological activity. Typically in the prior art to date, it is desiredto have the PEG stably linked to the protein so that the beneficialproperties provided by PEG remain. Another problem with some protein PEGconjugates is that upon decomposition of the conjugate undesirableproducts may be formed.

[0006] PEG has also been described for use in improving the bloodcirculation lifetime of liposomes (U.S. Pat. No. 5,103,556). Here, thePEG is covalently attached to the polar head group of a lipid in orderto mask or shield the liposomes from being recognized and removed by thereticuloendothelial system. Liposomes having releasable PEG chains havealso been described, where the PEG chain is released from the liposomeupon exposure to a suitable stimulus, such as a change in pH(PCT/US97/18813). However, release of the PEG chain from the liposomesuffers from the drawback that the decomposition products are chemicallymodified and can have unpredictable, potentially negative effects invivo.

SUMMARY OF THE INVENTION

[0007] Accordingly, it is an object of the invention to provide acompound where a ligand is covalently yet reversibly linked to ahydrophilic polymer. Upon cleavage of the linkage, the ligand in itsnative form is regenerated.

[0008] In one aspect, the invention includes a compound having thegeneral structure:

[0009] wherein R¹ is a hydrophilic polymer comprising a linkage forattachment to the dithiobenzyl moiety; R² is selected from the groupconsisting of H, alkyl and aryl; R³ is selected from the groupconsisting of O(C═O)R⁴, S(C═O)R⁴, and O(C═S)R⁴; R⁴ comprises anamine-containing ligand; and R⁵ is selected from the group consisting ofH, alkyl and aryl; and where orientation of CH₂—R³ is selected from theortho position and the para position.

[0010] In one embodiment, R⁵ is H and R² is selected from the groupconsisting of CH₃, C₂H₅ and C₃H₈. In another embodiment, R² and R⁵ arealkyls.

[0011] In another embodiment, the amine-containing ligand R⁴ is selectedfrom the group consisting of a polypeptide, an amine-containing drug andan amine-containing lipid. In an embodiment where the amine-containingligand R⁴ is an amine-containing lipid, the lipid includes either asingle hydrocarbon tail or a double hydrocarbon tail. In one preferredembodiment, the lipid is a phospholipid having a double hydrocarbontail.

[0012] The hydrophilic polymer R¹ can be, in yet another embodiment,selected from the group consisting of polyvinylpyrrolidone,polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,polyhydroxypropyloxazoline, polyhydroxypropyl-methacrylamide,polymethacrylamide, polydimethyl-acrylamide,polyhydroxypropylmethacrylate, polyhydroxyethylacrylate,hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol,polyaspartamide, copolymers thereof, and polyethyleneoxide-polypropyleneoxide.

[0013] In one preferred embodiment, the hydrophilic polymer R¹ ispolyethyleneglycol. In another embodiment, when R¹ is polyethyleneglycol, R⁵ is H and R² is CH₃ or C₂H₅.

[0014] In still another embodiment, the amine-containing ligand R⁴ is apolypeptide. The polypeptide can be, in another embodiment, arecombinant polypeptide. Exemplary and preferred polypeptides includecytokines, such as interferons, interleukins, and growth factors, andenzymes.

[0015] In another aspect, the invention includes a compositioncomprising a conjugate obtainable by reaction with a compound having thegeneral structural formula:

[0016] wherein R¹ is a hydrophilic polymer comprising a linkage forattachment to the dithiobenzyl moiety; R² is selected from the groupconsisting of H, alkyl and aryl; R³ is selected from the groupconsisting of O(C═O)R⁴, S(C═O)R⁴, and O(C═S)R⁴; R⁴ comprises a leavinggroup; and R⁵ is selected from the group consisting of H, alkyl andaryl; and where orientation of CH₂—R³ is selected from the orthoposition and the para position. The composition also includes apharmaceutically-acceptable carrier, such as saline, buffer or the like.

[0017] In one embodiment of this aspect, R² is selected from the groupconsisting of CH₃, C₂H₅ and C₃H₈.

[0018] In another embodiment, R³ is O(C═O)R⁴ and R⁴ is a hydroxy- oroxy-containing leaving group. The leaving group, in another embodiment,is derived from a compound selected from the group consisting ofchloride, para-nitrophenol, ortho-nitrophenol,N-hydroxy-tetrahydrophthalimide, N-hydroxysuccin imide,N-hydroxy-glutarimide, N-hydroxynorbornene-2,3-dicarboxyimide,1-hydroxybenzotriazole, 3-hydroxypyridine, 4-hydroxypyridine,2-hydroxypyridine, 1-hydroxy-6-trifluoromethylbenzotriazole, immidazole,triazole, N-methyl-imidazole, pentafluorophenol, trifluorophenol andtrichlorophenol.

[0019] In one embodiment, the claimed compound is reacted with anamine-containing ligand that displaces R⁴ to form a conjugate thatincludes the amine-containing ligand. For example, the amine-containingligand can be a phospholipid.

[0020] In a preferred embodiment, the hydrophilic polymer R¹ ispolyethyleneglycol, R⁵ is H and R² is CH₃ or C₂H₅.

[0021] In yet another aspect of this embodiment, the compositioncontaining the conjugate comprises a liposome. The liposome can furthercomprise an entrapped therapeutic agent.

[0022] In another embodiment, the amine-containing ligand comprises apolypeptide.

[0023] In yet another aspect, the invention includes a liposomecomposition comprising liposomes which include a surface coating ofhydrophilic polymer chains wherein at least a portion of the hydrophilicpolymer chains have the general structure:

[0024] wherein R¹ is a hydrophilic polymer comprising a linkage forattachment to the dithiobenzyl moiety; R² is selected from the groupconsisting of H, alkyl and aryl; R³ is selected from the groupconsisting of O(C═O)R⁴, S(C═O)R⁴, and O(C═S)R⁴; R⁴ comprises anamine-containing ligand; and R⁵ is selected from the group consisting ofH, alkyl and aryl; and where orientation of CH₂—R³ is selected from theortho position and the para position. The liposomes have a longer bloodcirculation lifetime than liposomes having hydrophilic polymer chainsjoined to the liposome via an aliphatic disulfide linkage.

[0025] In one embodiment, the liposome further comprises an entrappedtherapeutic agent.

[0026] In still another aspect, the invention includes a method forimproving the blood circulation lifetime of liposomes having a surfacecoating of releasable hydrophilic polymer chains. The method includespreparing liposomes that have between about 1% to about 20% of acompound having the general structure:

[0027] wherein R¹, R², R³, and R⁵ are as described above and R⁴comprises an amine-containing lipid.

[0028] In a preferred embodiment of this aspect, R⁵ is H and R² isselected from the group consisting of CH₃, C₂H₅ and C₃H₈.

[0029] In another embodiment, the amine-containing lipid comprises aphospholipid and R¹ is polyethyleneglycol.

[0030] In this aspect, the liposomes can further comprise an entrappedtherapeutic agent.

[0031] These and other objects and features of the invention will bemore fully appreciated when the following detailed description of theinvention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1A shows an embodiment of the invention where thedithiobenzyl (DTB) links a methoxy-polyethyelene glycol (mPEG) moietyand the amine-containing ligand;

[0033]FIG. 1B shows the products after thiolytic cleavage of thecompound in FIG. 1A;

[0034]FIG. 2 illustrates a synthetic reaction scheme for synthesis ofthe mPEG-DTB-amine-lipid, where the amine-ligand is the lipiddistearoylphosphatidylethanolamine (DSPE);

[0035]FIG. 3 illustrates the thiolytic cleavage mechanism of apara-dithiobenzyl urethane (DTB)-linked mPEG-DSPE conjugate;

[0036] FIGS. 4A-4B show a synthetic reaction scheme for preparation ofan mPEG-DTB-DSPE compound in accord with the invention where the DTBlinkage is sterically hindered by an alkyl group;

[0037]FIG. 5 shows another synthetic reaction scheme for preparation ofan mPEG-DTB-ligand compound in accord with the invention;

[0038]FIG. 6A is a synthetic reaction scheme for synthesis of anmPEG-DTB-lipid which upon thiolytic cleavage yields a cationic lipid;

[0039]FIG. 6B shows the products after thiolytic cleavage of thecompound in FIG. 6A;

[0040]FIG. 7A shows the rate of cleavage of ortho-mPEG-DTB-DSPE andpara-mPEG-DTB-DSPE conjugates in solution to form micelles in bufferalone (ortho-conjugate (*); para-conjugate (+)) and in the presence of150 μM cysteine (ortho-conjugate (open circles); para-conjugate (opensquares);

[0041]FIG. 7B shows the rate of cleavage of micellar mPEG-DTB-DSPEconjugates as described in FIG. 7A and of ortho-mPEG-DTB-DSPE (solidcircles) and para-mPEG-DTB-DSPE (solid squares) conjugates formulated inliposomes and incubated in the presence of 150 μM cysteine;

[0042] FIGS. 8A-8B show percentage of content release of entrappedfluorophore from liposomes comprised of DOPE:ortho-mPEG-DTB-DSPE (FIG.8A) or of DOPE:para-mPEG-DTB-DSPE (FIG. 8B) incubated in the presence ofcysteine at the indicated concentrations;

[0043]FIG. 9A shows normalized percent release of entrapped fluorophoreas a function of time for liposomes comprised of DOPE andpara-mPEG-DTB-DSPE. The percent release of entrapped fluorophore isnormalized with respect to percent release of fluorophore from liposomesincubated in the absence of cysteine. The release rate from liposomesincubated in the presence of cysteine at concentrations of 15 μM (solidsquares), 75 μM (open triangles), 150 μM (X symbols), 300 μM (opencircles), 1500 μM (solid circles), 3000 μM (+symbols), and 15000 μM(open diamonds) is shown;

[0044]FIG. 9B shows normalized percent release of entrapped fluorophoreas a function of time for liposomes comprised of DOPE andpara-mPEG-MeDTB-DSPE. The percent release of entrapped fluorophore isnormalized with respect to percent release of fluorophore from liposomesincubated in the absence of cysteine. The release rate for liposomesincubated in the presence of cysteine at concentrations of 15 μM (solidsquares), 75 μM (open triangles), 150 μM (X symbols), 300 μM (opencircles), 1500 μM (solid circles), 3000 μM (+symbols), and 15000 μM(open diamonds) is shown;

[0045]FIG. 9C shows normalized percent release of entrapped fluorophoreas a function of time for liposomes comprised of DOPE andmPEG-meDTB-distearoyl-glycerol compound of FIG. 6A. The percent releaseof entrapped fluorophore is normalized with respect to percent releaseof fluorophore from liposomes incubated in the absence of cysteine. Therelease rate of dye upon cleavage of the compound from liposomesincubated in the presence of cysteine at concentrations of 15 μM (solidsquares), 75 μM (open triangles), 150 μM (X symbols), 300 μM (opencircles), 1500 μM (solid circles), 3,000 μM (+symbols), and 15,000 μM(open diamonds) is shown;

[0046]FIG. 10 is a plot showing the amount of liposomes, in counts perminute/mL of liposomes containing entrapped In¹¹¹, in blood samplestaken from mice at various times after injection of liposomes comprisedof PHPC:cholesterol:mPEG-DTB-DSPE (55:40:5 molar ratio). One group ofanimals received a 200 μL injection of 200 mM cysteine at time zero(solid squares). The control group was injection with saline at timezero (open circles);

[0047]FIG. 11A shows a synthetic reaction scheme for synthesis of anmPEG-DTB-protein compound in accord with another embodiment of theinvention;

[0048]FIG. 11B shows the decomposition products after thiolytic cleavageof the compound in FIG. 11A;

[0049]FIG. 12 is a rendering of a photograph of ansodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE)profile of lysozyme reacted for 15 minutes (Lane 1) or for 1 hour (Lane2) with mPEG-MeDTB-nitrophyenylchloroformate to form amPEG-MeDTB-lysozyme conjugate, native lysozyme (Lane 3), lysozymereacted for 1 hour with mPEG-nitrophenylchloroformate (Lane 4),molecular weight markers (Lane 5), and the samples of Lanes 1-4 treatedwith 2% β-mercaptoethanol for 10 minutes at 70° C. (Lanes 6-9);

[0050]FIG. 13 shows the decomposition products after thiolytic cleavageof the an mPEG-DTB-p-nitroanilide conjugate;

[0051]FIG. 14A shows the absorbence as a function of wavelength, in nm,of mPEG-MeDTB-para-nitroanilide (closed diamonds) and after in vitroincubation with 5 mM cysteine for 2 minutes (closed squares), 5 minutes(x symbols), 10 minutes (open squares), 20 minutes (triangles), 40minutes (open diamonds) and 80 minutes (closed circles); and

[0052]FIG. 14B shows the amount of para-nitroanilide, in mole/L,released in vitro as a function of time, in minutes, frommPEG-MeDTB-para-nitroanilide conjugate incubated in the presence of 5 mMcysteine (closed circles), 1 mM cysteine (closed squares) and 0.15 mMcysteine (closed diamonds).

DETAILED DESCRIPTION OF THE INVENTION

[0053] I. Definitions

[0054] “Polypeptide” as used herein refers to a polymer of amino acidsand does not refer to a specific length of a polymer of amino acids.Thus, for example, the terms peptide, oligopeptide, protein, and enzymeare included within the definition of polypeptide. This term alsoincludes post-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations, and the like.

[0055] “Amine-containing” intends any compound having a moiety derivedfrom ammonia by replacing one or two of the hydrogen atoms by alkyl oraryl groups to yield general structures RNH₂ (primary amines) and R₂NH(secondary amines), where R is any hydrocarbyl group.

[0056] “Hydrophilic polymer” as used herein refers to a polymer havingmoieties soluble in water, which lend to the polymer some degree ofwater solubility at room temperature. Exemplary hydrophilic polymersinclude polyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline,polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropyl-methacrylamide, polymethacrylamide,polydimethyl-acrylamide, polyhydroxypropylmethacrylate,polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose,polyethyleneglycol, polyaspartamide, copolymers of the above-recitedpolymers, and polyethyleneoxide-polypropylene oxide copolymers.Properties and reactions with many of these polymers are described inU.S. Pat. Nos. 5,395,619 and 5,631,018.

[0057] “Polymer comprising a reactive functional group” or “polymercomprising a linkage for attachment” refers to a polymer that has beenmodified, typically but not necessarily, at a terminal end moiety forreaction with another compound to form a covalent linkage. Reactionschemes to functionalize a polymer to have such a reactive functionalgroup of moiety are readily determined by those of skill in the artand/or have been described, for example in U.S. Pat. No. 5,613,018 or byZalipsky et al., in for example, Eur. Polymer. J., 19(12):1177-1183(1983); Bioconj. Chem., 4(4):296-299 (1993).

[0058] “Recombinant” as in “recombinant polypeptide” implies joining ofamino acids through laboratory manipulation into a desired sequence.

[0059] “Alkyl” as used herein intends a group derived from an alkane byremoval of a hydrogen atom from any carbon atom: “C_(n)H_(2n+1)”. Thegroups derived by removal of a hydrogen atom from a terminal carbon atomof unbranched alkanes form a subclass of normal alkyl (n-alkyl) groups:H[CH₂]_(n). The groups RCH₂—, R₂CH—(R not equal to H), and R₃C—(R notequal to H) are primary, secondary and tertiary alkyl groupsrespectively.

[0060] “Aryl” refers to a substituted or unsubstituted monovalentaromatic radical having a single ring (e.g., benzene) or two condensedrings (e.g., naphthyl). This term includes heteroaryl groups, which arearomatic ring groups having one or more nitrogen, oxygen, or sulfuratoms in the ring, such as furyl, pyrrole, pyridyl, and indole. By“substituted” is meant that one or more ring hydrogens in the aryl groupis replaced with a halide such as fluorine, chlorine, or bromine; with alower alkyl group containing one or two carbon atoms; nitro, amino,methylamino, dimethylamino, methoxy, halomethoxy, halomethyl, orhaloethyl.

[0061] An “aliphatic disulfide” linkage intends a linkage of the formR′—S—S—R″, where R′ and R″ are linear or branched alkyl chains that maybe further substituted.

[0062] The following abbreviations are used herein: PEG, poly(ethyleneglycol); mPEG, methoxy-PEG; DTB, dithiobenzyl; MeDTB,methyl-dithiobenzyl, EtDTB, ethyl-dithiobenzyl; DSPE, distearoylphosphatidylethanolamine; DOPE, dioleoyl phosphatidylethanolamine; PHPC,partially hydrogenated phosphatidylcholine; MALDI-TOFMS, matrix-assistedlaser desorption/ionization time-of-flight mass spectrometry.

[0063] II. The Compound of the Invention

[0064] In one aspect, the invention comprises a compound of the form:

[0065] wherein R¹ comprises a hydrophilic polymer including functionalgroup suitable for covalently attaching the polymer to the dithiobenzylmoiety. R² and R⁵ are independently selected to be H, an alkyl or anaryl, and, as will be seen, can be varied to tailor the rate ofdisulfide cleavage. For example, to achieve a faster rate of cleavage,R² and R⁵ are hydrogen. A slower rate of cleavage is achieved bysterically hindering the disulfide by selecting an alkyl or aryl for oneor both of R² and R⁵. R³ comprises a linking moiety joined to R⁴, whichcomprises an amine-containing ligand. The linking moiety in preferredembodiments is O(C═O), S(C═O) or O(C═O). The amine-containing ligand R⁴can be a primary or a secondary amine and can be selected from anynumber of substrates, including, but not limited to lipids, drugs,polypeptides, viruses, surfaces of biomaterials and aminoglycosides. Inpreferred embodiments, R⁴ is a primary or secondary amine-containinglipid, drug or polypeptide. In the compound of the invention, theorientation of the group CH₂—R³ can be either ortho or para.

[0066]FIG. 1A shows the structure of an exemplary compound in accordwith the invention, where R¹ is the hydrophilic polymermethoxy-polyetheylene glycol, mPEG═CH₃O(CH₂CH₂O)_(n) where n is fromabout 10 to about 2300, which corresponds to molecular weights of about440 Daltons to about 100,000 Daltons. The molecular weight of thepolymer depends to some extent on the selection of R³. In embodimentswhere R³ is an amine-containing lipid for use in a liposome a preferredrange of PEG molecular weight is from about 750 to about 10,000 Daltons,more preferably from about 2,000 to about 5,000 Daltons. The mPEG inthis embodiment includes a urethane linking moiety. In embodiments whereR³ is an amine-containing polypeptide a preferred range of PEG molecularweight is from about 2,000 to about 40,000 Daltons, more preferably fromabout 2,000 to about 20,000 Daltons. It will be appreciated that R¹ canbe selected from a variety of hydrophilic polymers, and exemplarpolymers are recited above. It will also be appreciated that for someligands, such as polypeptides, the molecular weight of the polymer maydepend on the number of polymer chains attached to the ligand, where alarger molecular weight polymer is often selected when the number ofattached polymer chains is small.

[0067] With continuing reference to FIG. 1a, R² and R⁵ in this exemplarycompound are H, however either or both R² and R⁵ can also be a straightchain or branched alkyl or an aryl group. In a preferred embodiment, R⁵is H and R² is an alkyl, and several examples are given below. In thecompound shown in FIG. 1A, R³ takes the general form ofO(C═O)—(NH₂-ligand), where the NH₂-ligand can be any amine-containingpolypeptide, drug or lipid, and specific examples of each embodiment aregiven below. R3 can also be of the form O(C═S)—(NH₂-ligand) orS(C═O)—(NH₂-ligand).

[0068]FIG. 1B shows the mechanism of thiolytic cleavage of themPEG-DTB-(NH₂-ligand) compound of FIG. 1A. The ortho- orpara-dithiobenzyl carbamate moiety is cleavable under mild thiolyticconditions, such as in the presence of cysteine or othernaturally-occurring reducing agents. Upon cleavage, the amine-containingligand is regenerated in its natural, unmodified form. Studies insupport of the invention, described below, show that natural,physiologic conditions in vivo are sufficient to initiate and achievecleavage of the DTB linkage. It will be appreciated that a reducingagent can also be administered to artificially induce thiolyticconditions sufficient for cleavage and decomposition of the compound.

[0069] As noted above, R³ takes the general form of a linking moiety,such as O(C═O), S(C═O) or O(C═S) joined to an amine-containing ligand.In preferred embodiment, the amine-containing ligand comprises anamine-containing polypeptide, drug or lipid. Examples of theseembodiments will now be described.

[0070] A. Amine-Containing Lipid

[0071] In one embodiment, the amine-containing ligand is anamine-containing lipid. Lipids as referred to herein intendwater-insoluble molecules having at least one acyl chain containing atleast about eight carbon atoms, more preferably an acyl chain containingbetween about 8-24 carbon atoms. A preferred lipid is a lipid having anamine-containing polar head group and an acyl chain. Exemplary lipidsare phospholipids having a single acyl chain, such as stearoylamine, ortwo acyl chains. Preferred phospholipids with an amine-containing headgroup include phosphatidylethanolamine and phosphatidylserine. The lipidtail(s) can have between about 12 to about 24 carbon atoms and can befully saturated or unsaturated. One preferred lipid isdistearoylphosphatidylethanolamine (DSPE), however those of skill in theart will appreciate the wide variety of lipids that fall within thisdescription. It will also be appreciated that the lipid can naturallyinclude an amine group or can be derivatized to include an amine group.Other lipid moieties that do not have an acyl tail, such ascholesterolamine, are also suitable.

[0072] The synthesis of a polymer-DTB-lipid compound is schematicallydepicted in FIG. 2. mPEG derivatives (MW 2000 and 5000 Daltons) having amethoxycarbonyldithioalkyl end group were prepared by reacting2-(methoxycarbonyldithio)ethaneamine with mPEG-chloroformate, which wasreadily prepared by phosgenation of dried mPEG-OH solution (Zalipsky,S., et al., Biotechnol. Appl. Biochem. 15:100-114 (1992).). The formercompound was obtained through 2-aminoethanethiol hydrochloride reactionwith an equivalent amount of methoxycarbonylsulfenyl chloride, accordingto published procedures (Brois, S. J., et al., J. Amer. Chem. Soc.92:7629-7631 (1970); Koneko, T., et al., Bioconjugate Chem. 2:133-141(1991)). Both the para and ortho isomers of mercaptobenzyl alcohol(Grice, R., et al., J. Chem. Soc. 1947-1954 (1963)) coupled cleanly withthe resulting PEG-linked acyldisulfide, yielding mPEG bearing a dithiobenzyl alcohol end group. Active carbonate introduction proceeded aswith underivatized mPEG-OH, to give the para-nitrophenyl carbonate.Addition of DSPE in ethanolamine formed the desired mPEG-DTB-DSPEproduct. Both ortho- and para-DTB-lipid compounds were prepared andpurified by silica gel chromatography and characterized by NMR andMALDI-TOFMS, the details of which are given in Example 1.

[0073]FIG. 3 shows the mechanism of thiolytic cleavage of themPEG-DTB-DSPE conjugate. Upon cleavage, the phosphatidylethanolaminelipid is regenerated in its natural, unmodified form.

[0074] FIGS. 4A-4B show a reaction scheme for synthesis of mPEG-DTB-DSPEconjugates having an alkyl group adjacent the disulfide linkage, e.g., amore hindered disulfide linkage. As described more fully in Example 2A,mPEG-OH in dichloromethane was reacted with p-nitrophenylchloroformatein the presence of triethylamine (TEA) to form mPEG-nitrophenylcarbonate. An amino alcohol, such as 1-amino-2-propanol or1-amino-2-butanol, in dimethylformamide (DMF) was reacted with themPEG-nitrophenyl carbonate in the presence of TEA to form a secondaryalcohol attached to PEG. The secondary alcohol was then converted to thedesired mPEG-DTB-DSPE compound as illustrated in FIG. 4A and detailed inExample 2A.

[0075] In this reaction scheme, mPEG-methyl-dithiobenzyl- nitrophenylchloroformate was reacted with DSPE to form the desired compound. Thenitrophenyl chloroformate moiety in themPEG-methyl-dithiobenzyl-nitrophenyl chloroformate compound acts as aleaving group to yield the desired product upon reaction with a selectedlipid. The invention contemplates, in another aspect, a composition thatcomprises a compound produced by reaction with a compound such asmPEG-methyl-dithiobenzyl-R³, where R³ represents a leaving group joinedthrough a linking moiety to the benzene ring. The leaving group isdisplaced upon reaction with an amine-containing ligand, such as DSPE, apolypeptide or an amine-containing drug. The leaving group is selectedaccording to the reactivity of the amine in the ligand, and ispreferably derived from various acidic alcohols that have a hydroxy- oroxy-containing leaving group. These include chloride, p-nitrophenol,o-nitrophenol, N-hydroxy-tetrahydrophthalimide, N-hydroxysuccinimide,N-hydroxy-glutarimide, N-hydroxynorbornene-2,3-dicarboxyimide,1-hydroxybenzotriazole, 3-hydroxypyridine, 4-hydroxypyridine,2-hydroxypyridine, 1-hydroxy-6-trifluoromethylbenzotriazole, immidazole,triazole, N-methyl-imidazole, pentafluorophenol, trifluorophenol andtrichlorophenol.

[0076] Example 2B describes preparation of an mPEG-EtDTB-lipid conjugatewhere the disulfide linkage is hindered by an ethyl moiety.

[0077]FIG. 5 shows another synthetic reaction scheme for preparation ofan mPEG-DTB-ligand compound in accord with the invention. The details ofthe reaction procedure are given in Examples 3A-3B. Briefly, cold1-amino-2-propanol was reacted with sulfuric acid to form2-amino-1-methylethyl hydrogen sulfate. This product was reacted withcarbon disulfide and sodium hydroxide in aqueous ethanol to yield5-methylthiazolidine-2-thione. An aqueous solution of hydrochloric acidwas added to the 5-methylthiazolidine-2-thione and heated. Afterrefluxing for one week, the product, 1-mercapto(methyl)ethyl ammoniumchloride, was crystallized and recovered. This product was reacted withmethoxy carbonylsulfenyl chloride to yield2-(methoxycarbonyldithio)ethaneamine. Reaction of the2-(methoxycarbonyldithio)ethaneamine with mPEG-chloroformate using theprocedure described above with respect to FIG. 2 yields the desiredmPEG-DTB-nitrophenyl compound suitable for reaction with a selectedamine-containing ligand to form a compound in accord with the invention.

[0078] Example 3B describes the reaction for synthesis ofmPEG-(ethyl)DTB-nitrophenyl.

[0079]FIG. 6A shows a reaction scheme for preparation of anothermPEG-DTB-lipid compound in accord with the invention. The reactiondetails are provided in Example 4. The lipid 1,2-distearoyl-sn-glycerolis activated for reaction with mPEG-DTB-nitropheynl, prepared asdescribed in FIG. 4A or FIG. 5. The resulting mPEG-DTB-lipid differsfrom the compounds described above in the absence of a phosphate headgroup. The mPEG-DTB-lipid of FIG. 6A is neutral prior to cleavage. Asshown in FIG. 6B, upon thiolytic reduction of the disulfide bond, thecompound decomposes to yield a cationic lipid. The positively-chargedlipid provides for electrostatic interaction in vivo and commensurateadvantages in in vivo targeting.

[0080] In the reaction schemes described above, R⁵ of the claimedcompound is H. However, in other embodiments R⁵ is an alkyl or an arylmoiety. In this approach, for example where R² and R⁵ are both CH₃moieties, an α, β-unsaturated acyl chloride (R′R″C═CHCOCl, where R′ is,for example CH₃ and R″ is CH₃, however any alkyl or aryl iscontemplated) is reacted with an amine-terminated PEG to give thecorresponding N-PEG-substituted α, β-unsaturated amide. This compound isreacted with thiolacetic acid, giving the correspondingN-PEG-substituted β-(acetylthio) amide via conjugate addition to the C═Cbond. The acetylthio group (—SCOCH₃) is hydrolyzed to a thiol group(—SH), which is then reacted with methyl (chlorosulfenyl)formate(CISCOOCH₃), generating a methoxycarbonyl diothio group (—SSCOOCH₃);this intermediate is then reacted with p-mercapto benzyl alcohol to givethe N-PEG-substituted β-(dithiobenzyl alcohol) amide (having thestructure PEG-NH—CO—CH₂CR′R″—SS-p-phenyl—CH₂OH). The benzyl alcoholmoiety is then reacted with nitrophenyl chloroformate to give thenitrophenyl carbonate leaving group, as above.

[0081] 1. In vitro Cleavage of mPEG-DTB-DSPE Compound

[0082] The in vitro rate of cleavage of ortho-mPEG-DTB-DSPE andpara-mPEG-DTB-DSPE (prepared as described in Example 1) was studied bypreparing micellar solutions of the compounds in a buffered aqueoussolution (pH 7.2). Thiolytic cleavage of the compounds was monitored inthe presence and absence of 150 μM cysteine by analyzing fordisappearance of the compounds by HPLC, as described in Example 5. Theresults are illustrated in FIG. 7A where the ortho- and para-compoundsin the absence of cysteine (* symbols and +symbols, respectively) showno cleavage and are stable under these conditions in the absence ofcysteine. The ortho- and para-compounds, represented by the open circlesand the open squares, respectively, in the presence of 150 μM cysteinecleave as shown in FIG. 7A. The ortho-compound exhibited a slightlyfaster rate of decomposition than its para counterpart (T_(1/2)≈12minutes vs. ≈18 minutes).

[0083] 2. Liposome Compositions Comprising an mPEG-DTB-lipid Compound

[0084] a). In vitro Characterization

[0085] In one embodiment, the mPEG-DTB-lipid compound is formulated intoliposomes. Liposomes are closed lipid vesicles used for a variety oftherapeutic purposes, and in particular, for carrying therapeutic agentsto a target region or cell by systemic administration of liposomes. Inparticular, liposomes having a surface coating of hydrophilic polymerchains, such as polyethylene glycol (PEG), are desirable as drugcarries, since these liposomes offer an extended blood circulationlifetime over liposomes lacking the polymer coating. The polymer chainsin the polymer coating shield the liposomes and form a “stiff brush” ofwater solvated polymer chains about the liposomes. Thus, the polymeracts as a barrier to blood proteins, preventing binding of the proteinand recognition of the liposomes for uptake and removal by macrophagesand other cells of the reticuloendothelial system.

[0086] Typically, liposomes having a surface coating of polymer chainsare prepared by including in the lipid mixture between about 1 to about20 mole percent of the lipid derivatized with the polymer. The actualamount of polymer derivatized lipid can be higher or lower depending onthe molecular weight of the polymer. In the present invention, liposomesare prepared by adding between about 1 to about 20 mole percent of thepolymer-DTB-lipid conjugate to other liposome lipid bilayer components.As will be demonstrated in the studies described below, liposomescontaining the polymer-DTB-lipid conjugate of the invention have a bloodcirculation lifetime the is longer than liposomes containing apolymer-lipid conjugate where the polymer and lipid are joined by acleavable aliphatic disulfide bond.

[0087] In studies performed in support of the invention, liposomescomprised of the vesicle-forming lipid partially hydrogenatedphosphatidyl choline along with cholesterol and the ortho-mPEG-DTB-DSPEor the para-mPEG-DTB-DSPE compound were prepared as described in Example6. Cysteine-mediated cleavage of the mPEG-DTB-DSPE compounds wasmonitored in the presence and absence of 150 μM cysteine in an aqueousbuffer. The results are shown in FIG. 7B, which includes the data ofFIG. 7A for comparison. In FIG. 7B, the ortho- and para-compounds inmicellar form in the absence of cysteine (* symbols and +symbols,respectively) show no cleavage, which indicates stability of theconjugate in the absence of thiols. The open circles and the opensquares correspond to the ortho- and para-compounds, respectively, inmicellar form in the presence of cysteine, as discussed above withrespect to FIG. 7A. The solid circles and the solid squares correspondto the ortho- and para-compounds, respectively, in liposomal form in thepresence of cysteine.

[0088] The data in FIG. 7B shows that both the ortho and para compoundwere slightly more resistant to thiolytic cleavage when incorporatedinto liposomes. Examination of the thiolysis reaction products by TLC(silica gel G, chloroform/methanol/water 90:18:2) (Dittmer, J. C., etal., J. Lipid Res. 5:126-127 (1964)) showed DSPE as the sole lipidcomponent and another spot corresponding to a thiol-bearing, lipid-freemPEG derivative.

[0089] In another study performed in support of the invention, liposomeswere prepared from the lipid dioleoyl phosphatidylethanolamine (DOPE)and either the ortho-mPEG-DTB-DSPE or the para-mPEG-DTB-DSPE compoundwere prepared. DOPE is a hexagonal phase lipid which alone does not formlipid vesicles. However, liposomes will form when DOPE is combined witha few mole percent of the mPEG-DTB-DSPE compound. Cleavage of themPEG-DTB-DSPE compound triggers decomposition of the liposomes andrelease of liposomally-entrapped contents. Thus, the content releasecharacteristics of such liposomes provides for a convenient quantitativeevaluation of cleavable PEG-bearing liposomes.

[0090] Liposomes comprised of DOPE and the ortho- or para-mPEG-DTB-DSPEcompound were prepared as described in Example 7A with entrappedfluorophores, p-xylene-bis-pyridinium bromide and trisodium8-hydroxypyrenetrisulfonate. Release of the fluorophores from liposomesincubated in the presence of cysteine at various concentrations wasmonitored as described in Example 7B.

[0091] Results for liposomes comprising the ortho-compound are shown inFIG. 8A, where percentage of content release of entrapped fluorophorefrom liposomes incubated in the presence of cysteine at concentrationsof 15 μM (solid diamonds), 150 μM (solid inverted triangles), 300 μM(solid triangles) and 1.5 mM (solid circles) are shown. FIG. 8B is asimilar plot for liposomes comprising the para-compound, where theliposomes are incubated in cysteine at concentrations of 15 μM (soliddiamonds), 300 μM (solid triangles), 1 μM (solid squares) and 1.5 mM(solid circles).

[0092] FIGS. 8A-8B show that both the ortho- and para-compounds whenincorporated into liposome are cleaved, as evidenced by release of theentrapped dye, at a rate dependent on the concentration of cysteine.Control studies with non-cleavable mPEG-DSPE containing liposomesproduced no content release (results not shown here). These results alsosuggest that the ortho conjugate is somewhat more susceptible tothiolytic cleavage. For example, 300 μM cysteine liberates most of thecontents of DOPE liposomes within 20 minutes. Under the same conditions,only a fraction of liposomes having para-mPEG-DTB-DSPE decomposed.Similarly, after incubation for 20 minutes at 150 μM cysteine, half ofthe entrapped contents was released for the ortho-containing liposomes,while only approximately 10% of the contents were release in liposomescontaining the para-compound. Both ortho and para compounds havehalf-lives of less than 20 minutes at a cysteine level of 150 μM (seedata in FIG. 7B). This suggests that more than half of the originalthree mole percent of the mPEG-DTB-lipid must be cleaved to observecontent release from the liposomes.

[0093] Decomposition of the mPEG-DTB-DSPE/DOPE liposomes in 15 μMcysteine, the average plasma concentration in both humans and rodents(Lash, L. H., et al., Arch. Biochem. Biophys. 240:583-592 (1985)), wasminimal in the time frame of these experiments (60 minutes). Thissuggests that the mPEG-DTB-lipid compounds should have sufficiently longlifetimes in plasma to allow the PEG-grafted vesicles to distributesystemically in vivo, or to accumulate in a specific site eitherpassively or through ligand-mediated targeting. Local or short termincrease in cysteine concentration can potentially be achieved by itsintravenous or intra-arterial administration. The results shown in FIGS.8A-8B also suggest that a prolonged exposure to the natural plasmacysteine concentration (≈15 μM) would be sufficient to decompose most ofthese compounds. These suggestions were studied in in vivo experiments,described below.

[0094] In another study performed in support of the invention, liposomescomprised of DOPE and three different mPEG-DTB-lipid compounds wereprepared. The liposomes were prepared as described in Example 7 andincluded and entrapped fluorophore. The three mPEG-DTB-lipid compoundswere mPEG-DTB-DSPE as shown in FIG. 1A; mPEG-MeDTB-DSPE as shown in FIG.4B, where R is CH₃, and mPEG-MeDTB-distearoyl-glycerol, as shown in FIG.6A. The liposomes were comprised of 97 mole percent DOPE and 3 molepercent of one of the mPEG-DTB-lipid compounds. Cysteine-mediated rateof cleavage of the compounds was determined by monitoring the release ofentrapped fluorophore as a function of time in the presence of variouscysteine concentrations. The results are shown in FIGS. 9A-9C where thepercent release of entrapped fluorophore is normalized for the releaserate from liposomes incubated in buffer alone.

[0095]FIG. 9A shows the percent release of entrapped fluorophore as afunction of time for liposomes comprised of DOPE and para-mPEG-DTB-DSPE(compound of FIG. 1A). The release rate from liposomes containing theconjugate and incubated in the presence of cysteine at concentrations of15 μM (solid squares), 75 μM (open triangles), 150 μM (X symbols), 300μM (open circles), 1500 μM (solid circles), 3000 μM (+symbols), and15000 μM (open diamonds) is shown.

[0096]FIG. 9B shows the percent release of entrapped fluorophore as afunction of time for liposomes comprised of DOPE and paramPEG-MeDTB-DSPE (compound of FIG. 4B). The release rate of thefluorophore from liposomes incubated in the presence of cysteine atconcentrations of 15 μM (solid squares), 75 μM (open triangles), 150 μM(X symbols), 300 μM (open circles), 1500 μM (solid circles), 3000 μM(+symbols), and 15000 μM (open diamonds) is shown.

[0097]FIG. 9C is a similar plot for liposomes formed with DOPE andmPEG-MeDTB-distearoyl glycerol (compound of FIG. 6A). The release rateof dye from liposomes incubated in the presence of cysteine atconcentrations of 15 μM (solid squares), 75 μM (open triangles), 150 μM(X symbols), 300 μM (open circles), 1500 μM (solid circles), 3000 μM(+symbols), and 15000 μM (open diamonds) is shown.

[0098] FIGS. 9A-9C show that the rate of mPEG-MeDTB-lipid cleavage iscysteine-concentration dependent, with a slow rate of cleavage, asevidenced by release of entrapped fluorophore, at cysteineconcentrations of 15-75 μM. In comparing the data in FIG. 9A with thatin FIG. 9B, it is seen that the mPEG-MeDTB-DSPE compound (FIG. 9B)cleaves approximately 10 times more slowly than the mPEG-DTB-DSPEcompound (FIG. 9A). Thus, the rate of cleavage can be tailored accordingto the R moiety (see FIG. 2) in the DTB linkage.

[0099] b). In vivo Characterization

[0100] The blood circulation lifetime of liposomes prepared as describedin Example 8 and that include a polymer-DTB-lipid conjugate in accordwith the invention was determined in mice. In¹¹¹ was entrapped in theliposomes and the liposomes were administered by intravenous injection.One group of test animals additionally received an injection ofcysteine, the control group of animals additionally received aninjection of saline. Blood samples were taken at various times andanalyzed for the presence of liposomes, as evidenced by the presence ofIn¹¹¹.

[0101]FIG. 10 shows the results where the counts per minute (CPM) ofIn¹¹¹ is shown as a function of time following injection of theliposomes and saline (open circles) or 200 mM cysteine (solid squares).As seen, the cleavage of the mPEG-DTB-DSPE occurred upon exposure to thenaturally-occurring physiologic conditions, as evidenced by the cleavagein the group of mice treated with saline after administration of theliposomes. Administration of an exogeneous reducing agent, cysteine, tothe mice was effective to increase the rate of cleavage of themPEG-DTB-lipid compound in the time frame from between about 2 hours toabout 8 hours.

[0102] Importantly, cleavage of the polymer-DTB-lipid compound of theinvention results in regeneration of the original lipid in unmodifiedform. This is desirable since unnatural, modified lipids can haveundesirable in vivo effects. At the same time, the compound is stablewhen stored in the absence of reducing agents.

[0103] In other studies, not shown here, the blood circulation lifetimeof liposomes containing the mPEG-DTB-lipid were compared to liposomescontaining a polymer-lipid conjugate where the polymer and lipid arejoined by a cleavable aliphatic disulfide bond. Aliphatic disulfidelinkages are readily cleaved in vivo and the blood circulation lifetimeof liposomes having polymer chains grafted to their surface by analiphatic disulfide typically do not have the extended blood circulationlifetime observed for liposomes having stably linked polymer chains. Thedithiolbenzyl linkage of the invention, and in particular the morehindered DTB linkages, are more stable in vivo and achieve a longerblood circulation lifetime than liposomes with polymer chains attachedvia an aliphatic disulfide linkage.

[0104] B. Amine-Containing Polypeptide

[0105] In another embodiment, the invention includes a compound asdescribed with respect to FIG. 1A, where the amine-containing ligand isa polypeptide. A synthetic reaction scheme showing preparation of apolymer-DTB-polypeptide is shown in FIG. 11A, with mPEG as the exemplarypolymer. In general, a mPEG-DTB-leaving group compound is preparedaccording to one the synthetic routes described above in FIGS. 2, 4A and5. The leaving group can be nitrophenyl carbonate or any one of theothers described above. The mPEG-DTB-nitrophenyl carbonate compound iscoupled to an amine moiety in a polypeptide by a urethane linkage. The Rgroup adjacent the disulfide in the compound can be H, CH₃, C₂H₅ or thelike and is selected according to the desired rate of disulfidecleavage.

[0106]FIG. 11B shows the decomposition products upon cysteine-mediatedcleavage of the compound. As seen the native protein with nomodification to the protein amine group is regenerated upon cleavage.

[0107] Attachment of polymer chains, such as PEG, to a polypeptide oftendiminishes the enzymatic or other biological activity, e.g., receptorbinding, of the polypeptide. However, polymer modification of apolypeptide increases the blood circulation lifetime of the polypeptide.In the present invention, the polymer-modified polypeptide isadministered to a subject. As the polymer-modified polypeptidecirculates exposure to physiologic reducing conditions, such as bloodcysteine and other in vivo thiols, initiates cleavage of the polymerchains from the polypeptide. As the polymer chains are released from thepolypeptide, the biological activity of the polypeptide is graduallyrestored. In this way, the polypeptide initially has a sufficient bloodcirculation lifetime for biodistribution, and over time regains its fullbiological activity as the polymer chains are cleaved.

[0108] In a study performed in support of the invention, lysozyme wasused as a model polypeptide and an mPEG-MeDTB-lysozyme conjugate wasprepared by a synthetic route similar to those described above. Lysozymewas incubated with mPEG-MeDTB -nitrophenylcarbonate in 0.1 M borate, atpH 9 at a 2:1 ratio of nitrophenylcarbonate to amino group of lysozyme.After reactions times of 15 minutes and 3 hours, samples werecharacterized by SDS-PAGE. A comparative compound was prepared byreacting lysozyme under the same conditions for 60 minutes with aconjugate of mPEG-nitrophenyl carbonate, which will form a stablemPEG-lysozyme conjugate.

[0109]FIG. 12 shows a rendering of the SDS-PAGE gel. Lane 1 correspondsto the compound formed after 15 minutes reaction of lysozyme withmPEG-MeDTB-nitrophyenylcarbonate and Lane 2 represents the compoundformed after a 1 hour reaction time of the same compounds. Lane 3represents native lysozyme and Lane 4 corresponds to lysozyme reactedfor 1 hour with mPEG-nitrophenylcarbonate. The molecular weight markersin Lane 5 are as follows, from the top down: Molecular Weight (kDaltons)Marker 1163 β-galactosidase 97.4 phosphorylase b 66.3 bovine serumalbumin 55.4 glutamic dehydrogenase 36.5 lactate dehydrogenase 31carbonic anhydrase 21.5 trypsin inhibitor 14.4 lysozyme

[0110] Comparison of Lane 1 and Lane 2 shows that the longer reactiontime results in an increase in compound molecular weight, consistentwith additional mPEG chains conjugated to the polypeptide at longerincubation time.

[0111] Lanes 6-9 of the SDS-PAGE profile correspond to the samples inLanes 1-4 after treatment with 2% β-mercaptoethanol for 10 minutes at70° C. The mPEG-MeDTB-lysozyme conjugate after exposure to a reducingagent decomposed to regenerate native lysozyme, as evidenced by the bandin Lanes 6 and 7 at 14.4 kDa. In contrast, the stable mPEG-lysozmecompound was not affected upon incubation with a reducing agent, asevidenced by the agreement in the profile in Lane 9 and Lane 4.

[0112] Also evident from the SDS-PAGE profile is that covalentattachment of mPEG-MeDTB to a protein forms a mixture of conjugatescontaining various mPEG-protein ratios. This ratio is dependent on thereaction time and conditions. This is clearly seen in viewing the bandsin Lanes 1 and 2, where Lane 1 shows lysozyme derivatized with fromabout 1-6 PEG chains. In Lane 2, the longer reaction time yieldedmPEG-MeDTB-lysozyme conjugates with a higher mPEG-protein ratio. Allcleavable conjugates were readily cleaved to regenerate the nativeprotein, as seen in the bands of Lanes 6 and 7.

[0113] It will be appreciated that any of the hydrophilic polymersdescribed above are contemplated for use. The molecular weight of thepolymer is selected depending on the polypeptide, the number of reactiveamines on the polypeptide and the desired size of the polymer-modifiedcompound.

[0114] Polypeptides contemplated for use are unlimited and can benaturally-occurring or recombinantly produced polypeptides. Small, humanrecombinant polypeptides are preferred, and polypeptides in the range of10-30 KDa are preferred. Exemplary polypeptides include cytokines, suchas tumor necrosis factor (TNF), interleukins and interferons,erythropoietin (EPO), granulocyte colony stimulating factor (GCSF),enzymes, and the like. Viral polypeptides are also contemplated, wherethe surface of a virus is modified to include one or more polymer chainlinked via a DTB reversible linkage. Modification of a virus containinga gene for cell transfection would extend the circulation time of thevirus and reduce its immunogenicity, thereby improving delivery of anexogeneous gene.

[0115] C. Amine-Containing Drug

[0116] In yet another embodiment of the invention, a compound of theform polymer-DTB-amine-containing drug is contemplated. The compound isof the structure described above, and in particular with respect to FIG.1A where the amine-containing ligand in the figure is theamine-containing drug. Modification of therapeutic drugs with PEG iseffective to improve the blood circulation lifetime of the drug and toreduce any immunogenicity.

[0117] A polymer-DTB-amine-containing drug is prepared according to anyof the reaction schemes described above, with modifications as necessaryto provide for the particular drug. A wide variety of therapeutic drugshave a reactive amine moiety, such as mitomycin C, bleomycin,doxorubicin and ciprofloxacin, and the invention contemplates any ofthese drugs with no limitation. It will be appreciated that theinvention is also useful for drugs containing an alcohol or carboxylmoiety. In the case where the drug contains a hydroxyl or carboxylmoiety suitable for reaction, the polymer-DTB moiety can be linked tothe drug via urethane, ester, ether, thioether or thioester linkages. Inall of these embodiments, the polymer-DTB-drug compound afteradministration in vivo thiolytically decomposes to regenerate theamine-containing drug in its native, active form, therapeutic activityof the compound after modification and prior to administration is notnecessary. Thus, in cases where modification of the drug with theDTB-polymer causes a reduction or loss of therapeutic activity, afteradministration and cleavage of the DTB-polymer from the drug, activityof the drug is regained.

[0118] In studies performed in support of the invention, the drugnitroanilide was reacted with mPEG-MeDTB-nitrophenylcarbonate to form anmPEG-MeDTB-para-nitroanilide compound, as shown in FIG. 13.Decomposition of the compound upon exposure to a reducing agent yieldsthe products shown in the figure, with the drug para-nitroanilideregenerated in an unmodified state.

[0119] The mPEG-MeDTB-para-nitroanilide compound was incubated in vitroin buffer containing 5 mM cysteine and the absorbence of sampleswithdrawn at various times is shown in FIG. 14A. Seen in the figure aresamples measured at the following time points: time zero (closeddiamonds), 2 minutes (closed squares), 5 minutes (x symbols), 10 minutes(open squares), 20 minutes (triangles), 40 minutes (open diamonds) and80 minutes (closed circles). The change in the UV spectra as a functionof incubation time in cysteine is evident, showing cysteine-mediatedrelease of para-nitroanilide from the mPEG-MeDTB- para-nitroanilidecompound.

[0120]FIG. 14B shows the amount of para-nitroanilide, in mole/L,released in vitro from the mPEG-MeDTB-para-nitroanilide conjugateincubated in the presence of 5 mM cysteine (closed circles), 1 mMcysteine (closed squares) and 0.15 mM cysteine (closed diamonds). Therate of drug release from the conjugate was dependent on theconcentration of reducing agent present.

[0121] From the foregoing, it can be seen how various objects andfeatures of the invention are met. The compounds of the inventioncomprise an amine-containing ligand reversibly joined to a hydrophilicpolymer via an ortho or para-disulfide of a benzyl urethane linkage.This linkage when subjected to mild thiolytic conditions is cleaved toregenerate the original amine-containing ligand in its unmodified form.The rate of cleavage can be controlled by steric hinderance of thedisulfide in the linkage and/or by controlling the thiolytic conditionsin vivo. The compounds prior to cleavage of the dithiobenzyl linkage areprovided with an increased blood circulation lifetime, improvedstability and reduced immunogenicity.

III. EXAMPLES

[0122] The following examples further illustrate the invention describedherein and are in no way intended to limit the scope of the invention.

[0123] Materials

[0124] All materials were obtained from commercially suitable vendors,such as Aldrich Corporation.

Example 1 Synthesis of mPEG-DTB-DSPE

[0125] mPEG-MeDTB-nitrophenylcarbonate (300 mg, 0.12 mmol, 1.29 eq) wasdissolved in CHC1₃ (3 ml). DSPE (70 mg, 0.093 mol) and TEA (58.5 μl,0.42 mmol, 4.5 eq) were added to PEG-solution, and was stirred at 50° C.(oil bath temp). After 15 minutes, TLC showed that the reaction didn'tgo to completion. Then two portions of TEA (10 μl, and 20 μl), and fewportions of mPEG-MeDTB-nitrophenylcarbonate (50 mg, 30 mg, 10 mg) wereadded every after 10 minutes, until the reaction went to completion.Solvent was evaporated. Product mixture was dissolved in MeOH, and 1 gof C8 silica was added. Solvent was evaporated again. Product containingC8 silica was added on the top of the column, and was eluted withMeOH:H₂O gradient (pressure), MeOH:H₂O=30:70, 60 ml; MeOH:H₂O=50:50, 60ml; MeOH:H₂O═70:30, 140 ml (starting material eluted); MeOH:H₂O=75:25=40ml; MeOH:H₂O═80:20, 80 ml (product eluted); MeOH:H₂O=85:15, 40 ml;MeOH:H₂O=90:10, 40 ml; MeOH=40 ml; CHC1₃: MeOH:H₂O=90:18:10, 40 ml.Fractions containing pure product were combined and evaporated to giveproduct as colorless thick liquid. Tertiary butanol (5 ml) was added toit, lyophilized and the dried in vacuo over P₂O₅ to give product aswhite fluffy solid (252 mg, 89% yield).

[0126] The ortho- and para-DTB-DSPE compounds were purified by silicagel chromatography (methanol gradient 0-10% in chloroform, ≈70% isolatedyield) and the structures confirmed by NMR and MALDI-TOFMS. (¹H NMR forpara conjugate: (d6-DMSO, 360 MHz) δ 0.86 (t, CH₃, 6H), 1.22 (s, CH₂ oflipid, 56H), 1.57 (m, CH₂CH₂CO₂, 4H), 2.50 (2 xt, CH₂CO₂, 4H), 2.82 (t,CH₂S, 2H), 3.32 (s, OCH₃, 3H), 3.51 (m, PEG, ≈180H), 4.07 (t,PEG-CH₂OCONH, 2H), 4.11 & 4.28 (2×dd CH₂CH of glycerol, 2H), 4.98 (s,benzyl—CH₂, 2H), 5.09 (m, CHCH₂ of lipid), 7.35 & 7.53 (2×d, aromatic,4H) ppm. The ortho conjugate differed only in benzyl and aromaticsignals at 5.11 (s, CH₂, 2H), and 7.31 (d, 1H), 7.39 (m, 2H) 7.75(d, 1H)ppm.

[0127] MALDI-TOFMS produced a distribution of ions spaced at equal 44 Daintervals, corresponding to the ethylene oxide repeating units. Theaverage molecular weights of the compounds was 3127 and 3139 Da for paraand ortho isomers respectively (theoretical molecular weight ≈3100 Da).

[0128] The reaction scheme is illustrated in FIG. 2.

Example 2 Synthesis of mPEG-DTB-DSPE

[0129] A. mPEG-MeDTB-DSPE

[0130] This reaction scheme is illustrated in FIGS. 4A-4B.

[0131] mPEG(5K)—OH (40 g, 8 mmol) was dried azeotropically with toluene(total volume was 270 ml, 250 ml was distilled off by Dean-Stark).Dichloromethane (100 ml) was added to mPEG-OH. P-nitrophenylchloroformate (2.42 g, 12 mmol, 1.5 eq), and TEA (3.3 ml, 24 mmol, 3 eq)were added to PEG solution at 4° C. (ice water), while takingprecautions against moisture. Light yellow TEA hydrochloride salt wasformed. After 15 minutes cooling bath was removed, and the reactionmixture was stirred at room temperature overnight. TLC showed (CHC1₃:MeOH:H₂O=90:18:2) that the reaction was complete. Solvent wasevaporated. The residue was dissolved in ethyl acetate (˜50° C.). TEAhydrochloride salt was filtered off and washed with warm ethyl acetate.Solvent was evaporated and the product recrystallized with isopropanol(three times). Yield: 38.2 g (92%). ¹H NMR (DMSO-d₆, 360 MHz) δ 3.55 (s,PEG, 450H); 4.37 (t, PEG-CH₂, 2H); 7.55 (d, C₆H₅, 2H); 8.31 (d, C₆H₅,2H).

[0132] 1-Amino-2-propanol (1.1 ml, 14.52 mmol, 3 eq), and TEA (2.02 ml,14.52 mmol, 3 eq) were added to mPEG (5K)-nitrophenyl carbonate (25 g,4.84 mmol) in DMF (60 ml) and CH₂Cl₂ (40 ml). It was a yellow clearsolution. The reaction mixture was stirred at room temperature for 30minutes. TLC (CHC1₃: MeOH=90:10) showed that the reaction went tocompletion. Solvent (dichloromethane) was evaporated. Isopropanol (250ml) was added to the product mixture in DMF (60 ml). Productprecipitated immediately, and then recrystallized with iPrOH (threetimes). Yield: 22.12 g (90%). ¹H NMR (DMSO-d₆, 360 MHz) δ 0.98 (d,CH₃CH(OH)CH₂, 3H); 3.50 (s, PEG, 180H); 4.03 (t, PEG-CH₂, 2H); 4.50 (d,CH₃CHOH, 1H); 7.0 (t, mPEG-OCONH).

[0133] mPEG(5K)-urethane-2-methyl propanol (22.12 g, 4.34 mmol) wasdried azeotropically with toluene (45 ml). Dichloromethane (60 ml) wasadded to it. Methane sulfonyl chloride (604.6 μl, 7.81 mmol, 1.8 eq) andTEA (3.93 ml, 28.21 mmol, 6.5 eq) were added to mPEG-solution at 0° C.while maintaining stirring and taking precautions against moisture.After 30 minutes, cooling bath was removed, and the reaction mixture wasstirred at room temperature for 16 h. Solvent was evaporated. Ethylacetate was added to remove TEA salts. The product was recrystallizedwith isopropanol (three times). Yield: 20.27 g (90%). ¹H NMR (DMSO-d₆,360 MHz) δ 1.27 (d, CH₃CHOSO₂CH₃, 3H); 3.162 (s, CH₃O₂SOCH, 3H); 3.50(s, PEG, 180H); 4.07 (t, PEG-CH₂, 2H); 4.64 (q, CH₃CHOH, 1H); 7.43 (t,mPEG-OCONH).

[0134] mPEG(5K)-urethane-2methyl-methane sulfone (10.27 g, 1.98 mmol)was dried azeotropically with toluene (20 ml, each time). Sodium hydride(377 mg, 9.4 mmol, 4.75 eq) was added in anhydrous toluene (60 ml) at 0°C. (in ice water). After 5 minutes, triphenylmethanethiol (3.92 g, 14.6mmol, 7.15 eq) was added to the solution. After 10 minutes,mPEG-urethane-2methyl-methane sulfone (10.27 gm, 1.98 mmol) was added tothe reaction mixture. It became a yellow solution. After 45 minutes, TLC(CHC1₃: MeOH:H₂O=90:18:2) showed that the reaction went to completion.Acetic acid (445.57 μl, 7.42 mmol, 3.75 eq) was added to the reactionmixture to neutralize excess of sodium hydride. The solution becamethick and whitish. Solvent was evaporated and the solid wasrecrystallized with ethyl acetate (30 ml) and isopropanol (70 ml). Theproduct mixture did not dissolve completely, while precipitate filteredoff. Then the product mixture was recrystallyzed withisopropanol/tert-butyl alcohol (100 ml/20 ml). Yield: 8.87 g (84%). ¹HNMR (DMSO-d₆, 360 MHz) δ 0.74 (d, CH₃CHSC(C₆H₅)₃, 3H), 3.50 (s, PEG,180H), 4.0 (t, PEG-CH₂, 2H), 4.64 (q, CH₃CHOH, 1H); 7.49 (t,mPEG-OCONH); 7.20-7.41 (m, SC(C₆H₅)₃, 15H).

[0135] mPEG(5K)-urethane-2methyl-triphenylmethanethiol (8.87 g, 1.65mmol) was dissolved in TFA/CH₂C1₂ (10 ml/10 ml) at 0° C. Under vigorousstirring, methoxy carbonylsulfenyl chloride (185.5 μl, 1.99 mmol, 1.2eq) was added to the solution. The reaction mixture was stirred at roomtemperature for 15 minutes. TLC (CHCl₃: MeOH=90:10) showed that thereaction was complete. Solvents were evaporated. The product mixture wasrecrystallized with isopropanol:tert-butyl alcohol (80 ml: 20 ml) twotimes. Tertiary butanol (5 ml) was added to the product, which was thenlyophilized and dried in vacuo over P₂O₅ to give product as white fluffysolid (8.32 g, 97% yield). ¹H NMR (DMSO-d₆, 360 MHz) δ 1.17 (d,CH₃CHSSCOOCH₃, 3H); 3.42 (s, PEG, 180H); 3.84 (s, CH₃OCOSSCH, 3H); 4.05(t, mPEG-CH₂, 2H); 7.38 (t, mPEG-OCONH,1H).

[0136] mPEG(5K)-urethane ethyl(methyl)dithiocarbonyl methoxide (8.32 g,1.6 mmol) was dissolved in dry methanol (20 ml), and chloroform (2.5ml). A solution of mercapto benzyl alcohol (592 mg, 4 mmol, 2.5 eq) indry methanol (2 ml) was added to the PEG-solution. The reaction mixturewas stirred at room temperature for 18 h. Solvent was evaporated,product mixture was recrystallized with ethyl acetate/isopropanol, 30ml/100 ml (3 times). NMR showed ˜16% product was formed. So, anotherportion of mercapto benzyl alcohol (322 mg, 2.18 mmol, 1.8 eq) in MeOH(2 ml) was added dropwise to the product mixture in MeOH/CHCl₃ (24 ml/lml) at 0° C. (ice water). After addition (˜10 minutes) completion, icebath was removed, and the reaction mixture was stirred at roomtemperature for 24 h. TLC (CHC1₃: MeOH:H₂O=90:18:2) showed that thereaction was complete. Solvent was evaporated, and then product mixturewas recrystallized with ethyl acetate/isopropanol, 30 ml/100 ml. Yield:7.25 g, (94%). ¹H NMR (DMSO-d₆, 360 MHz) δ 1.56 (d, CH₃CHSSC₆H₅CH₂OH,3H); 3.29 (CH₃O-PEG, 3H); 3.50 (s, PEG, 450H); 4.03 (t, mPEG-CH₂, 2H);4.46 (d, HOCH₂C₆H₅, 2H); 5.16 (t, HOCH₂C₆H₅, 1H); 7.30 (d, C₆H₅, 2H);7.40 (br t, mPEG-OCONH, 1H); 7.50 (d, C₆H₅, 2H).

[0137] mPEG(5K)-urethane-ethyl(methyl)-dithiobenzyl alcohol (6.75 g,1.27 mmol) was dissolved in CHCl₃ (30 ml), P-nitrophenyl chloroformate(513 mg, 2.54 mmol, 2 eq) was added to it at 0° C. (ice water). After 5minutes triethylamine (531 μl, 3.81 mmol, 3 eq) was added. After 30minutes ice bath was removed, and the reaction mixture was stirred atroom temperature overnight. Solvent was evaporated. The product mixturewas dissolved in ethyl acetate. TEA salt was filtered off, and thensolvent was evaporated. Then the product mixture was recrystallized withethyl acetate/isopropanol, 30 ml/100 ml (three times). Yield: 6.55 g(94%). ¹H NMR (DMSO-d₆, 360 MHz) δ 1.17 (d, CH₃CHSSC₆H₅, 3H); 3.24(CH₃O-PEG, 3H); 3.40 (s, PEG, 180H); 4.03 (br t, mPEG-CH₂, 2H); 5.28 (S,C₆H₅CH₂OCO, 2H); 7.45-8.35 (m, C₆H₅)₂, 8H)

[0138] mPEG-MeDTB-nitrophenylcarbonate (766 mg, 0.14 mmol, 1.29 eq) wasdissolved in CHC1₃ (5 ml). DSPE (70 mg, 0.093 mol) and TEA (58.5 μl,0.42 mmol, 4.5 eq) were added to PEG-solution, and was stirred at 50° C.(oil bath temp). After 20 minutes, TLC showed that the reaction didn'tgo to completion. More mPEG-MeDTB-nitrophenylcarbonate (total 1239 mg,0.23 mmol, 2.47 eq) and 1-hydroxybenztriazole (HOBt) (25 mg, 0.19 mmol,2 eq) were added. After 20 minutes, TLC (CHC1₃: MeOH:H₂O=90:18:2, withmolybdenum and ninhydrin) showed that the reaction was complete. Solventwas evaporated. Product mixture was dissolved in warm (42° C.) ethylacetate. It was a cloudy solution (TEA salt precipitated). The solutionwas filtered, and solvent was evaporated. MeOH, and 2 g of C8 silica wasadded to the product mixture. Solvent was evaporated again. Productcontaining C8 silica was added on the top of the column, and was elutedwith MeOH:H₂O gradient (pressure), MeOH:H₂O 30:70, 100 ml; MeOH H₂O50:50, 100 ml; MeOH H₂O 70:30, 250 ml (starting material eluted); MeOHH₂O 75:25=40 ml; MeOH H₂O 80:20, 200 ml (product eluted); MeOH=100 ml;CHC1₃: MeOH:H₂O=90:18:2, 100 ml; CHC1₃: MeOH H₂O=75:36:6, 100 ml.Fractions containing pure product were combined and evaporated to giveproduct as colorless thick liquid. Tertiary butanol (5 ml) was added toit, lyophilized and then dried in vacuo over P₂O₅ to give product aswhite fluffy solid (467 mg, 83% yield). ¹H NMR (DMSO-d₆, 360 MHz) δ 0.83(d, 2(CH₃), 3H); 1.16 (d, CH₃CHSSC₆H₅, 3H); 1.21 (s, 28(CH₂, 56H); 1.47(br m, CH₂CH₂CO, 4H); 2.23 (2×t, CH₂CH₂CO, 4H); 3.50 (s, PEG, 180H);4.04 (br t, mPEG-CH₂, 2H); 4.05 (trans d, PO₄CH₂CHCH₂, 1H); 4.24 (cis d,PO₄CH₂CHCH₂, 1H); 4.97 (s, C₆H₅CH₂OCO -DSPE, 2H); 5.03 (br s, (PO₄CH₂CH,1H); 7.32 (d, C₆H₅, 2H); 7.53 (d, C₆H₅, 2H); 7.52 (br s, mPEG-OCONH,1H). MALDI-TOFMS produced a bell shaped distribution of ions spaced atequal 44 Da intervals, corresponding to the ethylene oxide repeatingunits. The average molecular mass of the conjugate and mPEG-thiol(mostly cleaved disulfide) is 6376 and 5368 Da (theoretical molecularmass ˜6053, and 5305 Daltons).

[0139] B. mPEG-ethylDTB-DSPE

[0140] mPEG-urethane ethyl(ethyl)dithiocarbonyl methoxide (2 g, 0.90mmol) was dissolved in dry methanol (8 ml). At the beginning thesolution was cloudy, but after 5 minutes it became a clear solution.Mercaptobenzyl alcohol (265.2 mg, 1.79 mmol, 2 eq) was added to thePEG-solution. The reaction mixture was stirred at room temperature for30 hours. Ether (70 ml) was added to the reaction solution toprecipitate the product, and kept at 4° C. overnight. The white solidwas filtered and recrystallized with ethyl acetate/ether, 30 ml/70 ml.Yield: 1.96 g, (94%). ¹H NMR (DMSO-d₆, 360 MHz) δ 0.86 (d,CH₃CH₂CHSSC₆H₅CH₂OH, 3H); 1.42 (p, CH₃CH₂CHSSC₆H₅CH₂OH, 1H); 1.64 (p,CH₃CH₂CHSSC₆H₅CH₂OH, 1H); 3.51 (s, PEG, 180H); 4.03 (t, mPEG-CH₂, 2H);4.47 (d, HOCH₂C₆H₅, 2H); 5.20 (t, HOCH₂C₆H₅, 1H); 7.31 (d, C₆H₅, 2H);7.42 (br t, mPEG-OCONH, 1H); 7.49 (d, C₆H₅, 2H).

[0141] N-hydroxy-s-norbornene-2,3-dicarboxylic acid imide (HONB) (48 mg,0.269 mmol) was added to DSPE (55 mg, 0.073 mmol) in CHCl₃ (3 ml) at 50°C. (oil bath temperature). After 3-4 minutes it became a clear solution.Then mPEG-EtDTB-nitrophenylchloroformate (334 mg, 0.134 mmol) was added,followed by triethylamine (TEA, 45 μl, 0.329 mmol). After 20 minutes TLC(CHCl₃:MeOH:H₂O=90:18:2) showed that the reaction went to completion(molybdenum and ninhydrin sprays). Solvent was evaporated. Productmixture was dissolved in methanol, mixed with C8 silica (1 g) andstriped of the solvent by rotary evaporation. The solid residue wasadded on the top of the C8-column, which was then eluted with MeOH:H₂Ogradient (pressure), MeOH:H₂O=30:70, 60 ml; MeOH:H₂O=50:50, 60 ml;MeOH:H₂O=70:30, 140 ml; MeOH:H₂O=75:25=140 ml (starting materialeluted); MeOH: H₂O=80:20, 80 ml; MeOH:H₂O=90:10, 140 ml (producteluted); MeOH=40 ml; CHCl₃:MeOH:H₂O=90:18:10, 40 ml. Fractionscontaining pure product were combined and evaporated to give product ascolorless thick liquid. Tertiary butanol (5 ml) was added, lyophilizedand then dried in vacuo over P₂O₅ to give product as white fluffy solid(175 mg, 78% yield). ¹H NMR (DMSO-d₆, 360 MHz) δ 0.85 (d, 2(CH₃), 6H; d,CH₃CHSSC₆H₅, 3H); 1.22 (s, 28(CH₂), 56H); 1.49 (br m, CH₂CH₂CO, 4H);2.24 (2×t, CH₂CH₂CO, 4H); 3.50 (s, PEG, 180H); 4.04 (br t, mPEG-CH₂,2H); 4.08 (trans d, PO₄CH₂CHCH₂, 1H); 4.27 (cis d, PO₄CH₂CHCH₂, 1H);4.98 (s, C₆H₅CH₂OCO-DSPE, 2H); 5.06 (br s, (PO₄CH₂CH, 1H); 7.34 (d,C₆H₅, 2H); 7.53 (d, C₆H₅, 2H); 7.55 (br s, mPEG-OCONH, 1H).

Example 3 Synthesis of mPEG-DTB-nitrophenylchloroformate

[0142] A. Procedures for synthesis of 1-(mercaptomethyl) ethylammoniumchloride

[0143] 1. 2-Amino-l-methylethyl hydrogen sulfate. 1-Amino-2-propanol(22.53 g, 0.3 mol) was vigorously stirred in an ice bath. Sulfuric acid(16.10 ml, 0.3 mol) was added very slowly, over the course of one hour.Thick vapors and a very viscous solution were formed in the flask. Afteraddition was complete, the reaction was heated between 170° C. and 180°C., under reduced pressure, connected to the house vacuum. Upon heating,the reaction turned light brown. After all water was removed(approximately 1 hour) it was allowed to cool to room temperature. Uponcooling a brown, glassy solid was formed which would crystallize whentriturated with methanol. It was dissolved in water (50 ml) at 60° C.Enough warm methanol was added to make the solution 80% methanol. Uponcooling, crystals formed which were then filtered and dried over P₂O₅.Yield: 17.17 g (37%). ¹H NMR (D₆-DMSO): δ 1.16 (d, CH₃, 3H); δ 2.78 (dd,NH₃—CH₂, 1H); δ 2.97 (dd, NH₃—CH₂, 1H); δ 4.41 (m, CH—OSO₃, 1H); δ 7.69(s, H₃N, 3H). Melting point: 248°-250° C. (lit: 250° C.)

[0144] 2. 5-Methylthiazolidine-2-thione. 2-Amino-1-methylethyl hydrogensulfate (23.03 g, 148 mmol) and carbon disulfide (10.71 ml, 178 mmol,1.2 eq.) were stirred in a 250 ml round-bottom-flask in 50% aqueousethanol (40 ml). To this, sodium hydroxide (13.06 g, 327 mmol, 2.2 eq.)in 50% aqueous ethanol (50 ml) was added drop-wise, very slowly. Uponaddition of sodium hydroxide, all starting materials dissolved and thesolution turned orange. The reaction was refluxed (85° C.) for 40minutes, after which time it turned bright yellow and a thickprecipitate was formed. Ethanol was evaporated and then the aqueoussolution was warmed and then filtered through a Buchner funnel to removeall water-soluble impurities. The remaining crystals were dissolved inwarm ethanol and then warm water was added until the solution was 80%water. The mixture was allowed to cool and then refrigerated, yieldinglong, needle-like crystals. Yield: 14.64 g (75%). ¹H NMR (D₆-DMSO): δ1.33 (d, CH₃, 3H); δ 3.50 (m, R₃CH, 1H); δ 3.95 (dd, N—CH₂, 1H); δ 4.05(m, N-CH₂, 1H); δ 10.05 (s, NH, 1H). Melting point: 92.5-93.5 (lit:94-95).

[0145] 3. 1-(mercaptomethyl)ethylammonium chloride.5-Methylthiazolidine-2-thione (6.5 g, 49 mmol) was placed in a 250 mlround-bottom-flask. A solution of aqueous hydrochloric acid (40 ml, 18%in H₂O) was added and the flask was heated in an oil bath. The reactionrefluxed (120° C.) for one week. Three times throughout the week 1 ml ofconcentrated hydrochloric acid was added. The reaction was monitoredusing TLC with ethyl acetate as eluent. They were visualized using UV,ninhydrin, and iodine vapors. Through most of the week the reaction wasa heterogeneous mixture, with the starting material as oil which wasdenser than water. After one week the oil starting material was gone,although still visible on TLC. The reaction was removed from heat andallowed to cool to room temperature, and then was refrigerated tocrystallize starting material. The crystallized starting material wasfiltered. Filtrate was evaporated and it was dried over P₂O₅ and NaOH toremove all water and HCl. The crude product was washed with two portionsof diethyl ether (50 ml each) to remove all starting material. It wasagain dried over P₂O₅. Yield: 2.83 g (45%). ¹H NMR (D₆-DMSO): δ 1.33 (d,CH₃, 3H); δ 2.92 (m, N—CH₂, 2H); δ 3.12 (m, SH, 1H); δ 3.18 (m, R₃—CH,1H); δ 8.23 (bs, NH₃, 3H). Melting point: 80-82° C. (lit: 92-94).

[0146] The reaction scheme is illustrated in FIG. 5.

[0147] B. Synthesis of mPEG-ethyl-DTB-nitrophenylchloroformate

[0148] 1. 2-Amino-l-ethylethyl hydrogen sulfate. 1-Amino-2-butanol (15ml, 158 mmol) was vigorously stirred in a 100 ml round-bottom-flask inan ice bath. Sulfuric acid (8.43 ml, 158 mmol) was added very slowly,over the course of one hour. Thick vapors and a very viscous solutionwere formed in the flask. After addition was complete, the reaction washeated between 170° and 180° C., under reduced pressure, connected tothe house vacuum. Upon heating, the reaction turned light brown. Afterall water was removed (approximately 1 hour) it was allowed to cool toroom temperature. Upon cooling a brown, glassy solid was formed. It wasdissolved in hot water (50 ml) and then placed in the refrigeratorovernight. Upon cooling, crystals formed which were then filtered anddried over P₂O₅. Yield: 9.98 g (37%). ¹H NMR (D₆-DMSO): δ 0.87 (t, CH₃,3H); δ 1.51 (q, CH₃—CH₂, 2H); δ 2.82 (dd, NH₃—CH₂, 1H); δ 3.00 (dd,NH₃—CH₂, 1H); δ 4.21 (m, CH—OSO₃, 1H); δ 7.70 (s, H₃N, 3H).

[0149] 2. 5-Ethylthiazolidine-2-thione. 2-Amino-l-ethyl-ethyl hydrogensulfate (9.98 g, 59 mmol) and carbon disulfide (4.26 ml, 71 mmol, 1.2eq.) were stirred in a 100 ml round-bottom-flask in 50% aqueous ethanol(15 ml). To this, sodium hydroxide (5.20 g, 130 mmol, 2.2 eq.) in 50%aqueous ethanol (20 ml) was added drop-wise, very slowly. Upon additionof sodium hydroxide, all starting materials dissolved and the solutionturned orange. The reaction was refluxed (85° C.) for 40 minutes, afterwhich time it turned bright yellow and a thick precipitate was formed.Ethanol was evaporated and then the aqueous solution was warmed and thenfiltered through a Buchner funnel to remove all water-solubleimpurities. The remaining crystals were dissolved in warm ethanol andthen warm water was added until the solution was 80% water. The mixturewas allowed to cool and then refrigerated, yielding needle-likecrystals. Yield: 7.28 g (86%). ¹H NMR (D₆-DMSO): δ 0.88 (t, CH₃, 3H); δ1.66 (in, CH₃—CH₂, 2H); δ 3.58 (m, R₃CH, 1H); δ 3.93 (m, N—CH₂, 2H); δ10.06 (s, NH,1H). Melting point: 76-78° (lit: 76.6-76.9).

[0150] 3. 1-(mercaptoethyl)ethylammonium chloride.5-Ethylthiazolidine-2-thione (7.24 g, 50 mmol) was placed in a 250 mlround-bottom-flask. A solution of aqueous hydrochloric acid (45 ml, 18%in H₂O) was added and the flask was heated in an oil bath. Upon heating,the starting material melted, forming, all heterogeneous mixture. Thereaction refluxed (120° C.) for one week. Four times throughout the week1 ml of concentrated hydrochloric acid was added. The reaction wasmonitored using TLC with ethyl acetate as eluent. They were visualizedusing UV, ninhydrin, and iodine vapors. Throughout the week the reactionwas a heterogeneous mixture, with the starting material as oil which wasdenser than water. The reaction was removed from heat and allowed tocool to room temperature, and then was refrigerated to crystallizestarting material. The crystallized starting material was filtered.Filtrate was evaporated and it was dried over P₂O₅ and NaOH to removeall water and HCl. The crude product was washed with two portions ofdiethyl ether (50 ml each) to remove all starting material. It was againdried over P₂O₅. Yield: 3.66 g (52%). ¹H NMR (D₆-DMSO):

[0151] The reaction scheme is illustrated in FIG. 5.

Example 4 Synthesis of mPEG-DTB-lipid

[0152] 1, 2-distereoyl-sn-glycerol (500 mg, 0.8 mmol) was driedazeotropically with benzene (3 times). Para-nitrophenyl chloroformate(242 mg, 1.2 mmol, 1.5 eq), dimethylaminopyridine (DMAP) (10 mg, 0.08mmol, 0.1 eq), and TEA (334.5 μl, 2.4 mmol, 3 eq) were added to 1,2-distereoyl glycerol in CHC1₃ (5 ml). The reaction mixture was stirredat room temperature for 2 h. TLC (Toluene: ethyl acetate=7:3) showedthat the reaction was complete. Then the product mixture was extractedwith 10% citric acid to remove dimethylaminopyridine (DMAP), washed withacetonitrile (3 ml, 4 times) to remove excess of p-nitrophenylchloroformate. Pure product was dried in vacuo over P₂O₅. Yield: 557mg(88%). %). ¹H NMR (CHC1₃, 360 MHz) δ 0.88 (t, end CH₃, 6H); 1.25 (s,28×CH₂, 56H); 1.58 (m, CH₂CH₂CO, 4H); 2.34 (2 xt, CH₂CO, 4H); 4.22(trans d, CH₂OCOC₁₇H₃₅, 1H); 4.35 (m, OCOOCH₂CH, 2H); 4.51 (cis d,CH₂OCOC₁₇H₃₅, 1H); 5.37 (m, OCOOCH₂CH, 1H); 7.39 (d, C₆H₅, 2H); 8.28 (d,C₆H₅, 2H).

[0153] Ethylene diamine (42 μl, 0.63 mmol, 5 fold excess), and pyridine(200 μl, were added in CHC1₃ (1 ml). 2-disteroyl-sn-p-nitrophenylcarbonate (100 mg, 0.13 mmol) was dissolved in CHCl₃ (1 ml) and addeddropwise to ethylene diamine solution with a pastuer pipette at 0° C.(ice water) and continued overnight (16 h). TLC (CHC1₃: MeOH:H₂O90:18:2, and CHC1₃: MeOH=90:10) showed that the reaction was complete.Solvent was evaporated to remove pyridine. Then the product mixture wasdissolved in CHC1₃, loaded onto the column (Aldrich, Silica gel, 60°A,200-400 mesh), and eluted with CHC1₃: CH₃COCH₃, and CHC1₃: MeOHgradient, CHC1₃:CH₃COCH₃=90:10, 60 ml (upper spot eluted); CHC1₃:NeOH=90:10, 60 ml (product eluted). Fractions containing pure productwere combined and evaporated. Tert-butanol was added and dried in vacuoover P₂O₅. Yield: 64 mg (75%). ¹H NMR (DMSO-d₆, 360 MHz) δ 0.83 (t, endCH₃, 6H); 1.22 (s, 28×CH₂, 56H); 1.51 (m, CH₂CH₂CO, 4H); 2.25 (2 xt,CH₂CO, 4H); 2.83 (m, H₂NCH₂CH₂NH, 2H); 3.21 (m, H₂NCH₂CH₂NH, 2H);4.10-4.14 (m & cis d, COOCH₂CHCH₂, 4H); 5.17 (m, OCOOCH₂CH, 1H); 7.78(m, H₂NCH₂CH₂NH, 2H).

[0154] mPEG-MeDTB-nitrophenylchloroformate (400 mg, 0.162 mmol, 2.2 eq)was dissolved in CHCl₃ in (2 ml). 1,2-steroyl-sn-ethylene amine (51 mg,0.075 mmol) and TEA (37 μl, 0.264 mmol, 3.52 eq) were added to thesolution. Then the reaction mixture was stirred at 45° C. for 20minutes. TLC (CHC1₃: MeOH:H₂O=90:18:2, and CHC1₃: MeOH=90:10) showedthat the reaction went to completion. Solvent was evaporated. Theproduct mixture was dissolved in methanol. 2 g of C8 silica was addedand then solvent was evaporated. C8 silica containing product mixturewas added on the top of the C8 column ((Supelco, Supel clean. Lot no.SP0824), and was eluted with MeOH:H₂O gradient (pressure),MeOH:H₂O=60:40, 40 ml; MeOH:H₂O =70:30, 80 ml (starting materialeluted); MeOH:H₂O=80:20, 40 ml; MeOH:H₂O=90:10=20 ml; CHC1₃:MeOH:H₂O=5:80:15, 20 ml; CHC1₃: MeOH:H₂O=90:18:10, 40 ml (producteluted). Fractions containing pure product were combined and evaporatedto give product as colorless thick liquid. Tertiary butanol (5 ml) wasadded and the solution was lyophilized and then dried in vacuo over P₂O₅to give product as white solid (200 mg, 89% yield). ¹H NMR (DMSO-d₆, 360MHz) δδ 0.83 (t, end CH₃, 6H); 1.22 (s, 28×CH₂, 56H); 1.48 (m, CH₂CH₂CO,4H); 2.25 (2×t, CH₂CO, 4H); 3.10 (m, HNCH₂CH₂NH, 4H); 3.50 (s, PEG,180H); 4.04 (t, mPEG-CH₂, 2H); 4.09 (trans d, COOCH₂CHCH₂, 1H); 4.25(cis d, COOCH₂CHCH₂, 1H); 4.98 (s, C₆H₅CH₂OCO, 2H); 5.23 (m,COOCH₂CHCH₂, 1H); 7.18 (m, NHCH₂CH₂NH, 2H); 7.33 (d, C₆H₅, 2H); 7.38 (m,mPEG-OCONH, 1H); 7.52 (d, C₆H₅, 2H).

[0155] The reaction scheme is illustrated in FIG. 6A.

Example 5 In Vitro Cleavage of mPEG-DTB-DSPE Compound

[0156] Ortho-mPEG-DTB-DSPE and para- mPEG-DTB-DSPE (prepared asdescribed in Example 1) were added to a buffered aqueous solution (pH7.2) in the presence and absence of 150 μM cysteine. Disappearance ofthe conjugates was monitored by HPLC (Phenomenex C₈ Prodigy, 4.6×50 mmcolumn, detection at 277 nm, mobile phase methanol/water 95:5 with 0.1%trifluoroacetic acid at 1 mL/min). The results are illustrated in FIG.7A where the ortho-conjugate is represented by the open circles and thepara-conjugate by the open squares.

Example 6 In Vitro Cleavage of o- and p-mPEG-DTB-DSPE Compound inLiposomes

[0157] A. Liposome Preparation

[0158] The lipids partially hydrogenated phosphatidylcholine (PHPC),cholesterol and ortho- or para-mPEG-DTB-DSPE (prepared as described inExample 1, mPEG MW=1980 Daltons) were dissolved in a 95:5:3 molar ratio,respectively, in a suitable organic solvent, typicallycholorform/methanol in a 1:1 or 1:3 ratio. The solvent was removed byrotary evaporation to form a dried lipid film. The film was hydratedwith aqueous buffer to from liposomes that were sized via extrusion toan average diameter of 120 nm.

[0159] B. In vitro Characterization

[0160] The liposomes were incubated in phosphate buffered saline, pH7.2, containing 5 mM EDTA at 37° C. in the presence of 150 μM cysteine.Disappearance of the conjugates was monitored by HPLC (Phenomenex C₈Prodigy, 4.6×50 mm column, detection at 277 nm, mobile phasemethanol/water 95:5 with 0.1% trifluoroacetic acid at 1 mL/min). Resultsare shown in FIG. 7B where the liposomes comprising the ortho-conjugateare represented by the solid circles and liposomes comprising thepara-conjugate by the solid squares. The open circles and the opensquares correspond to ortho-mPEG-DTB-DSPE and para-mPEG-DTB-DSPE inmicellar form (discussed above in Example 5, FIG. 7A).

Example 7 In vitro Cleavage of o- and p-mPEG-DTB-DSPE Compound inLiposomes

[0161] A. Liposome Preparation

[0162] The lipids dioleoyl phosphatidylethanolamine (DOPE) and ortho- orpara -mPEG-DTB-DSPE (prepared as described in Example 1, mPEG MW=1980Daltons) were dissolved a 97:3 molar ratio in chloroform/methanol 1:1.The solvent was removed by rotary evaporation to form a dried lipidfilm. The lipid film was hydrated with an aqueous solution containing 30mM each of the fluorophores p-xylene-bis-pyridinium bromide andtrisodium 8-hydroxypyrenetrisulfonate. was hydrated with aqueous bufferto from liposomes that were sized via extrusion to an average diameterof 100 nm.

[0163] B. In vitro Characterization

[0164] The liposomes were incubated in HEPES buffer, pH 7.2, at 37° C.in the presence of cysteine at concentrations of 15 μM, 150 μM, 300 μMand 1.5 mM. Percent of released dye was determined as the increase insample fluorescence (λ_(em)=512 nm, λex=413 nm—pH-independent isobesticpoint) over that of the preincubation sample (zero release) normalizedto the increase in fluorescence obtained after lysis of preincubationsample with 0.2% Triton X-100 (100% release) (Kirpotin, D. et al., FEBSLetters, 388:115-118 (1996)). Results at various cysteine concentrationsfor liposome comprising the ortho-compound are shown in FIG. 8A and forthe for the para-compound are shown in FIG. 8B.

Example 8 In Vivo Characterization of Liposomes Comprising mPEG-DTB-DSPECompound

[0165] A. Liposome Preparation

[0166] The lipids partially hydrogenated phosphatidylcholine (PHPC),cholesterol and para-mPEG-DTB-DSPE (prepared as described in Example 1,mPEG MW=1980 Daltons) were dissolved in a 55:40:5 mole percent ratio,respectively, in an organic solvent. The solvent was removed by rotaryevaporation to form a dried lipid film. The film was hydrated withaqueous buffer containing diethylene triamine pentacetic acid (EDTA) toform liposomes. After downsizing the liposomes to an average diameter of120 nm unentrapped EDTA was removed and In¹¹¹ was added to the externalmedium. The liposomes were incubated for a time sufficient for In¹¹¹ tocross the lipid bilayer and chelate with EDTA.

[0167] B. In vivo Administration

[0168] Mice were divided into two study groups. The liposome compositiondescribed above was injected into all test animals. One group of thetest animals also received a 200 μL injection of 200 mM cysteine at 1, 3and 5 hours post liposome injection. The other test group received aninjection of saline at the same time points. Liposome content in theblood was determined by monitoring blood samples for In¹¹¹. The resultsare shown in FIG. 10.

[0169] Although the invention has been described with respect toparticular embodiments, it will be apparent to those skilled in the artthat various changes and modifications can be made without departingfrom the invention.

It is claimed:
 1. A compound having the general structure:

wherein R¹ is a hydrophilic polymer having a molecular weight of between440-100,000 Daltons and comprising a linkage for attachment to thedithiobenzyl moiety; R² is selected from the group consisting of H,alkyl and aryl; R³ is selected from the group consisting of O(C═O)R⁴,S(C═O)R⁴, and O(C═S)R⁴; R⁴ comprises an amine-containing ligand; and R⁵is selected from the group consisting of H, alkyl and aryl; and whereorientation of CH₂—R³ is selected from the ortho position and the paraposition.
 2. The compound of claim 1, wherein R⁵ is H and R² is selectedfrom the group consisting of CH₃, C₂H₅ and C₃H₈.
 3. The compound ofclaim 1, wherein the amine-containing ligand R⁴ is selected from thegroup consisting of a polypeptide, an amine-containing drug and anamine-containing lipid.
 4. The compound of claim 1, wherein theamine-containing ligand R⁴ is an amine-containing lipid comprisingeither a single hydrocarbon tail or a double hydrocarbon tail.
 5. Thecompound of claim 4, wherein the amine-containing lipid is aphospholipid having a double hydrocarbon tail.
 6. The compound of claim1, wherein R² and R⁵ are alkyls.
 7. The compound of claim 1, wherein R¹is selected from the group consisting of polyvinylpyrrolidone,polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,polyhydroxypropyloxazoline, polyhydroxypropyl-methacrylamide,polymethacrylamide, polydimethyl-acrylamide,polyhydroxypropylmethacrylate, polyhydroxyethylacrylate,hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol,polyaspartamide, copolymers thereof, and polyethyleneoxide-polypropyleneoxide.
 8. The compound of claim 1, wherein R¹ is polyethyleneglycol. 9.The compound of claim 8, wherein R⁵ is H and R^(2 xis CH) ₃ or C₂H₅. 10.A liposome comprising the compound of claim
 1. 11. A liposome comprisingthe compound of claim
 9. 12. The compound of claim 1, wherein theamine-containing ligand R⁴ is a polypeptide.
 13. The compound of claim12, wherein the polypeptide is a recombinant polypeptide.
 14. Thecompound of claim 7, wherein the polypeptide is a recombinantpolypeptide.
 15. The compound of claim 12, wherein the polypeptide is acytokine.
 16. The compound of claim 12, wherein the polypeptide isselected from the group consisting of interferons, interleukins, growthfactors, and enzymes.
 17. A composition comprising a conjugateobtainable by reaction with a compound having the general structuralformula:

wherein R¹ is a hydrophilic polymer having a molecular weight of between440-100,000 Daltons and comprising a linkage for attachment to thedithiobenzyl moiety; R² is selected from the group consisting of H,alkyl and aryl; R³ is selected from the group consisting of O(C═O)R⁴,S(C═O)R⁴, and O(C═S)R⁴; R⁴ comprises a leaving group; and R⁵ is selectedfrom the group consisting of H, alkyl and aryl; and where orientation ofCH₂—R³ is selected from the ortho position and the para position; and apharmaceutically-acceptable carrier.
 18. The composition of claim 17,wherein R² is selected from the group consisting of CH₃, C₂H₅ and C₃H₈.19. The composition of claim 17, wherein R³ is O(C═O)R⁴ and R⁴ is ahydroxy- or oxy-containing leaving group.
 20. The composition of claim17, wherein the leaving group is derived from a compound selected fromthe group consisting of chloride, para-nitrophenol, ortho-nitrophenol,N-hydroxy-tetrahydrophthalimide, N-hydroxysuccinimide,N-hydroxy-glutarimide, N-hydroxynorbornene-2,3-dicarboxyimide,1-hydroxybenzotriazole, 3-hydroxypyridine, 4-hydroxypyridine,2-hydroxypyridine, 1-hydroxy-6-trifluoromethylbenzotriazole, immidazole,triazole, N-methyl-imidazole, pentafluorophenol, trifluorophenol andtrichlorophenol.
 21. The composition of claim 17, wherein said compoundis reacted with an amine-containing ligand that displaces R⁴ to form aconjugate comprising said amine-containing ligand.
 22. The compositionof claim 21, wherein the amine-containing ligand comprises aphospholipid.
 23. The composition of claim 22, wherein R¹ is selectedfrom the group consisting of polyvinylpyrrolidone, polyvinylmethylether,polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropyl-methacrylamide, polymethacrylamide,polydimethylacrylamide, polyhydroxypropylmethacrylate,polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose,polyethyleneglycol, polyaspartamide, copolymers thereof, andpolyethyleneoxide-polypropylene oxide.
 24. The composition of claim 22,wherein R¹ comprises polyethyleneglycol.
 25. The composition of claim24, wherein R² is CH₃ or C₂H₅.
 26. The composition of claim 25, whereinthe composition containing the conjugate comprises a liposome.
 27. Thecomposition of claim 26, wherein the liposome further comprises anentrapped therapeutic agent.
 28. The composition of claim 21, whereinthe amine-containing ligand comprises a polypeptide.
 29. The compositionof claim 29, wherein R¹ is selected from the group consisting ofpolyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline,polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropyl-methacrylamide, polymethacrylamide,polydimethyl-acrylamide, polyhydroxypropylmethacrylate,polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose,polyethyleneglycol, polyaspartamide, copolymers thereof, andpolyethyleneoxide-polypropylene oxide.
 30. The composition of claim 28,wherein R¹ comprises polyethyleneglycol.
 31. The composition of claim30, wherein the polypeptide comprises a recombinant polypeptide.
 32. Thecomposition of claim 30, wherein the polypeptide comprises a cytokine.33. The composition of claim 30, wherein the polypeptide is selectedfrom the group consisting of interferons, interleukins, growth factors,and enzymes.
 34. A liposome composition comprising: liposomes comprisinga surface coating of hydrophilic polymer chains wherein at least aportion of the hydrophilic polymer chains have the general structure:

wherein R¹ is a hydrophilic polymer having a molecular weight of between440-100,000 Daltons and comprising a linkage for attachment to thedithiobenzyl moiety; R² is selected from the group consisting of H,alkyl and aryl; R³ is selected from the group consisting of O(C═O)R⁴,S(C═O)R⁴, and O(C═S)R⁴; R⁴ comprises an amine-containing ligand; and R⁵is selected from the group consisting of H, alkyl and aryl; and whereorientation of CH₂—R³ is selected from the ortho position and the paraposition, wherein the liposomes have a longer blood circulation lifetimethan liposomes having hydrophilic polymer chains joined to the liposomevia an aliphatic disulfide linkage.
 35. The composition of claim 34,wherein R² is selected from the group consisting of CH₃, C₂H₅ and C₃H₈.36. The composition of claim 34, wherein the amine-containing lipidcomprises a phospholipid.
 37. The composition of claim 36, wherein R¹ isselected from the group consisting of polyvinylpyrrolidone,polyvinylmethylether, polymethyloxazoline, polyethyloxazoline,polyhydroxypropyloxazoline, polyhydroxypropyl-methacrylamide,polymethacrylamide, polydimethyl-acrylamide,polyhydroxypropylmethacrylate, polyhydroxyethylacrylate,hydroxymethylcellulose, hydroxyethylcellulose, polyethyleneglycol,polyaspartamide, copolymers thereof, and polyethyleneoxide-polypropyleneoxide.
 38. The composition of claim 36, wherein R¹ comprisespolyethyleneglycol.
 39. The composition of claim 38, wherein R² is CH₃or C₂H₅.
 40. The composition of claim 34, wherein the liposome furthercomprises an entrapped therapeutic agent.
 41. A method for improving theblood circulation lifetime of liposomes having a surface coating ofreleasable hydrophilic polymer chains, comprising preparing liposomesthat include between about 1% to about 20% of a compound having thegeneral structure:

 wherein R¹ is a hydrophilic polymer having a molecular weight ofbetween 440-100,000 Daltons and comprising a linkage for attachment tothe dithiobenzyl moiety; R² is selected from the group consisting of H,alkyl and aryl; R³ is selected from the group consisting of O(C═O)R⁴,S(C═O)R⁴, and O(C═S)R⁴; R⁴ comprises an amine-containing ligand; and R⁵is selected from the group consisting of H, alkyl and aryl; and whereorientation of CH₂—R³ is selected from the ortho position and the paraposition.
 42. The method of claim 41, wherein R⁵ is H and R² is selectedfrom the group consisting of CH₃, C₂H₅ and C₃H₈.
 43. The method of claim41, wherein the amine-containing lipid comprises a phospholipid.
 44. Themethod of claim 43, wherein R¹ is selected from the group consisting ofpolyvinylpyrrolidone, polyvinylmethylether, polymethyloxazoline,polyethyloxazoline, polyhydroxypropyloxazoline,polyhydroxypropyl-methacrylamide, polymethacrylamide,polydimethyl-acrylamide, polyhydroxypropylmethacrylate,polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose,polyethyleneglycol, polyaspartamide, copolymers thereof, andpolyethyleneoxide-polypropylene oxide.
 45. The method of claim 43,wherein R¹ comprises polyethyleneglycol.
 46. The method of claim 45,wherein R² is CH₃ or C₂H₅.
 47. The method of claim 41, wherein theliposome further comprises an entrapped therapeutic agent.